Differences in DNA replication in diploid and transformed human cells were studied. Transformed cells lacked the capacity to discriminate against the incorporation of 5-bromodeoxyuridine into DNA in the presence of thymidine, because 1) they are defective in the excision of misincorporated bases and/or 2) different DNA polymerases are used. Repair synthesis induced by UV or MNNG was inhibited more effectively by novobiocin or nalidixic acid than either replicative synthesis or repair synthesis induced by methylmethane-sulfonate (MMS), suggesting that topoisomerase-mediated alteration of higher order DNA structure is required for DNA repair. However, cells could tolerate the disruption (by topoisomerase inhibitors) of such higher-order DNA structure for up to 24 hours and resume repair synthesis after UV. Replicative DNA synthesis in tumor cells defective in the repair of alkylation damage (Mer minus) was far more sensitive to MNNG than that in repair-proficient lines (Mer plus). Both Mer plus and Mer minus cells could incise DNA after MNNG treatment, but the Mer minus cells did so more slowly and did not restore their original DNA conformation, even after 24 hours.